Myosin light chain kinase, and myosin light chain phosphatase have been isolated from rabbit alveolar macrophages. The myosin has been highly purified and the kinase and phosphatase partially purified. The myosin, phosphorylated by the kinase, has an actin-activated ATPase activity that varies directly with the extent of phosphorylation of its 20,000 dalton myosin light chain. Maximum activity is achieved at one mole of phosphate per mole of 20,000 dalton light chain. The phosphatase is capable of dephosphorylating the phosphorylated light chain, which can then be rephosphorylated with the kinase. The kinase and phosphatase together constitute a regulatory system for controlling actin-myosin interactions and hence contractility in macrophages.